The Raf/MEK/ERK (extracellular signal-regulated kinase) kinase cascade is pivotal in transmitting signals from membrane receptors to transcription factors that control gene expression culminating in the regulation of cell cycle progression (Robinson, M J and Cobb, M H (1997) Curr. Opin. Cell Biol. 9:180-186). This cascade can prevent cell death through ERK2 and p90(Rsk) activation and phosphorylation of apoptotic and cell cycle regulatory proteins (Shelton J G et al (2003) Oncogene. 22(16):2478-92). The PI3K/Akt kinase cascade also controls apoptosis and can phosphorylate many apoptotic and cell cycle regulatory proteins. These pathways are interwoven as Akt can phosphorylate Raf (Rapidly growing Fibrosarcoma) and result in its inactivation, and Raf can be required for the anti-apoptotic effects of Akt. Raf is a key serine-threonine protein kinase which participates in the transmission of growth, anti-apoptotic and differentiation messages. These signals can be initiated after receptor ligation and are transmitted to members of the MAP kinase cascade that subsequently activate transcription factors controlling gene expression. Raf is a multigene family which expresses oncoprotein kinases: Raf-1, A-Raf and B-Raf (McCubrey J A. et al (1998) Leukemia. 12(12):1903-1929; Ikawa et al (1988) Mol. and Cell. Biol. 8(6):2651-2654; Sithanandam et al (1990) Oncogene 5:1775-1780; Konishi et al (1995) Biochem. and Biophys. Res. Comm. 216(2):526-534). All three Raf kinases are functionally present in certain human hematopoietic cells, and their aberrant expression can result in abrogation of cytokine dependency. Their regulatory mechanisms differ because C-Raf and A-Raf require additional serine and tyrosine phosphorylation within the N region of the kinase domain for full activity (Mason et al (1999) EMBO J. 18:2137-2148), and B-Raf has a much higher basal kinase activity than either A-Raf or C-Raf. The three Raf oncoproteins play critical roles in the transmission of mitogenic and anti-apoptotic signals. B-Raf has recently been shown to be frequently mutated in various human cancers (Wan et al (2004) Cell 116:855-867). Development of specific Raf inhibitors may prove efficacious in cancer therapy. The cytoplasmic serine/threonine kinase B-Raf and receptor tyrosine kinases of the platelet-derived growth factor receptor (PDGFR) family are frequently activated in cancer by mutations of an equivalent amino acid. Structural studies have provided important insights into why these very different kinases share similar oncogenic hot spots and why the PDGFR juxtamembrane region is also a frequent oncogenic target (Dibb N J (2004) Nature Reviews. Cancer. 4(9):718-27).
Transformation of normal melanocytes into melanoma cells is accomplished by the activation of growth stimulatory pathways, typically leading to cellular proliferation, and the inactivation of apoptotic and tumor suppressor pathways. Small molecule inhibitors of proteins in the growth stimulatory pathways are under active investigation, and their application to melanoma patients would represent a new treatment strategy to inhibit cell proliferation or induce cell death (Polsky D. (2003) Oncogene, 22(20):3087-91; Konopleva, M et al. (2003) Blood, 102(11):625a).
B-Raf encodes a Ras-regulated kinase that mediates cell growth and malignant transformation kinase pathway activation that controls cell growth and survival. Activation of the Ras/Raf/MEK pathway results in a cascade of events from the cell surface to the nucleus ultimately affecting cellular proliferation, apoptosis, differentiation and transformation. Raf is a downstream effector enzyme of Ras. When activated, Raf goes on to activate MEK1 and MEK2 kinases which in turn phosphorylate and activate ERK1 and ERK2 which translocate to the nucleus where they stimulate pathways required for translation initiation and transcription activation leading to proliferation (Sorbera et al. (2002) Drugs of the Future 27(12):1141-1147). Activating B-Raf mutations have been identified in 66% of melanomas and a smaller percentage of many other human cancers. B-Raf mutations also account for the MAP kinase pathway activation common in non-small cell lung carcinomas (NSCLCs), including V600E and other mutations identified as novel, altering residues important in AKT-mediated B-Raf phosphorylation which suggest that disruption of AKT-induced B-Raf inhibition can play a role in malignant transformation. Although >90% of B-Raf mutations in melanoma involve codon 600 (57 of 60), 8 of 9 B-Raf mutations reported to date in NSCLC are non-V600 (89%; P<10(−7)), strongly suggesting that B-Raf mutations in NSCLC are qualitatively different from those in melanoma; thus, there may be therapeutic differences between lung cancer and melanoma in response to Raf inhibitors (Karasarides et al. (2004) Oncogene 23(37):6292-6298; Bollag et al. (2003) Current Opinion in Invest. Drugs 4(12):1436-1441). Although uncommon, B-Raf mutations in human lung cancers may identify a subset of tumors sensitive to targeted therapy (Brose M S et al. (2002) Cancer Research 62(23):6997-7000; US 2005/267060).
Raf protein kinases are key components of signal transduction pathways by which specific extracellular stimuli elicit precise cellular responses in mammalian cells. Activated cell surface receptors activate ras/rap proteins at the inner aspect of the plasma membrane which in turn recruit and activate Raf proteins. Activated Raf proteins phosphorylate and activate the intracellular protein kinases MEK1 and MEK2. In turn, activated MEKs catalyze phosphorylation and activation of p42/p44 mitogen-activated protein kinase (MAPK). A variety of cytoplasmic and nuclear substrates of activated MAPK are known which directly or indirectly contribute to the cellular response to environmental change. In fact, B-Raf mutation has been shown to predict sensitivity to pharmacological MEK inhibition by small molecule inhibitors by limiting tumor growth in B-Raf mutant xenografts (Solit et al (2005) Nature Letters to Editor 6 Nov. 2005, doi:10.1038). Three distinct genes have been identified in mammals that encode Raf proteins; A-Raf, B-Raf and C-Raf (also known as Raf-1) and isoformic variants that result from differential splicing of mRNA are known. In particular, it has been suggested that B-Raf is the major Raf isoform activated by the neurotrophin, nerve growth factor (NGF), for NGF induced extracellular signaling by kinase activation (York et al. (2000) Mol. and Cell. Biol. 20(21):8069-8083).
Cancer chemotherapy drugs typically have a narrow therapeutic index, and often the responses produced are only just palliative as well as unpredictable. In contrast, targeted therapy that has been introduced in recent years is directed against cancer-specific molecules and signaling pathways and thus has more limited nonspecific toxicities. Tyrosine kinases are an esp. important target because they play an important role in the modulation of growth factor signaling (Arora et al. (2005) Jour. of Pharm. and Exp. Ther. 315(3):971-979). Small molecule inhibitors of tyrosine kinase compete with the ATP binding site of the catalytic domain of several oncogenic tyrosine kinases (Fabian et al. (2005) Nature Biotechnology 23(3):329-336). Several tyrosine kinase inhibitors (TKIs) have been found to have effective antitumor activity and have been approved or are in clinical trials, including imatinib mesylate (STI571; Gleevec), gefitinib (Iressa), erlotinib (OSI-1774; Tarceva), lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), sutent (SU11248), and leflunomide (SU101). TKIs are thus an important new class of targeted therapy that interfere with specific cell signaling pathways and thus allow target-specific therapy for selected malignancies. The Raf/MEK/ERK pathway is the focus of intense drug discovery efforts (Thompson et al. (2005) Current Opinion in Pharmacology 5(4):350-356; Sridhar et al (2005) Molecular Cancer Therapeutics 4(4):677-685).
Inhibitors of Raf kinases have been suggested for use in disruption of tumor cell growth and hence in the treatment of cancers, e.g. histiocytic lymphoma, lung adenocarcinoma, small cell lung cancer and pancreatic and breast carcinoma; and also in the treatment and/or prophylaxis of disorders associated with neuronal degeneration resulting from ischemic events, including cerebral ischemia after cardiac arrest, stroke and multi-infarct dementia and also after cerebral ischemic events such as those resulting from head injury, surgery and/or during childbirth (Strumberg et al. (2005) Onkologie 28(2):101-107). Sorafenib (NEXAVAR™; BAY-43-9006; Bayer and Onyx) is an oral cytostatic pan-kinase inhibitor, approved by the FDA for advanced renal cell carcinoma, and is being developed for the potential treatment of additional, various cancers (Ahmad et al. (2004) Clinical Cancer Res. 10(18, Pt. 2):6388S-6392S; Lee et al. (2003) Current Opinion in Invest. Drugs 4(6):757-763). Sorafenib prevents tumor growth by inhibition of tumor cell proliferation and tumor angiogenesis (Clark et al. (2005) Clinical Cancer Res. 11(15):5472-5480; Yu et al. (2005) Oncogene 24(46):6861-6869; Wilhelm et al. (2004) Cancer Res. 64(19):7099-7109).
Use of these targeted therapies is not without limitations such as the development of resistance and the lack of tumor response in the general population. The availability of newer inhibitors and improved patient selection will help overcome these problems in the future. There remains a significant need to develop Raf kinase inhibitors for the treatment of solid tumors